G protein-coupled receptors (GPCRs) form the largest gene family in the human genome and are the most successful biochemical targets of pharmaceutical drugs. For about 140 GPCRs the natural ligands are unknown and they are thus called "orphan" receptors. Identification of these natural ligands is the crucial step to start studying their function and holds the potential for development of novel therapeutic drugs. We have developed a strategy to isolate such natural ligands by expressing the cloned receptors in suitable cell lines and monitoring activation of the receptors after adding fractions of tissue extracts. This strategy will ultimately allow us to purify the active molecules and determine their structure. In this application, we propose to isolate, identify and characterize pharmacologically the natural ligands of two such orphan GPCRs which are structurally related to endothelin receptors. Following the isolation of the natural ligands, we will characterize their pharmacological effects, study their biosynthesis by cloning the precursor proteins that encode them and analyze their distribution in the brain and peripheral organs. These receptors do not bind the known endothelin peptides but have been conserved during evolution across various species, showing that they must serve an important function. Both receptors are highly expressed in the brain and one of them was recently identified as a substrate for parkin, the gene product which is mutated in an autosomal recessive form of Parkinson's disease. Identification of the natural ligands will enable us to study the physiological functions of these two orphan GPCRs under normal and pathological conditions.